Tuberculosis (TB) is responsible for substantial morbidity and mortality worldwide. It is estimated that there are 9 million new cases of active TB every year, representing both primary TB and reactivation of latent infection, resulting in 1.3 million deaths. Only a small fraction (5-10%) of individuals infected with Mycobacterium tuberculosis (Mtb) develop active TB, with the rest being latently infected. Persons with latent infection exhibit immune responsiveness to mycobacterial antigens and no clinical signs of disease but remain at risk of reactivation to active TB. HIV co-infection increases the reactivation risk to 10% annually. The estimated 2 billion people worldwide (11.2 million in the U.S.) with latent infection are a significant reservoir of potential active, infectious TB. Due to he lack of an effective vaccine and increasing prevalence of drug resistance, there is no clear path for control, and ultimately elimination, in countries where TB is endemic. Since one third of the world's population is infected with Mtb, there is a critical need for better vaccines, treatments, and methods to determine those at highest risk of reactivation. TB predominately affects the lungs where tuberculosis lesions (granulomas) form that consist of lymphocytes, activated macrophages and neutrophils, as well as Mtb bacilli. TB granulomas are heterogeneous in terms of cellular composition, even within the same individual and may influence the ability of antibiotics to effectively sterilize individual lesions. This lesional heterogeneity is recapitulatd in our cynomolgus macaque model of TB. While [18F]-labeled 2-deoxy-2-fluoro- D-glucose (FDG) PET/CT imaging has been helpful in serially tracking Mtb infection and disease, as well as in monitoring the response to drugs in the macaque model and in humans, it does not specifically target the immune cells that are the primary populations in TB granulomas. Having agents that target key immune cells present in TB-infected lungs (macrophages, T cells, and neutrophils) will help differentiate resolving from progressing lesions, which will be invaluable in monitoring therapeutic responses as well as shortening pre- clinical and clinical trials of novel vaccines and chemotherapeutics. We hypothesize that PET imaging agents targeting specific populations of immune cells will provide more detailed information about the composition of granulomas in Mtb-infected cynomolgus macaques, such as the levels of neutrophils, T cells and activated macrophages that are important for early diagnosis of activated disease and assessing the response to drug treatment. In Aim 1, we will determine the mechanism of uptake for 64Cu-LLP2A, a VLA-4 targeted tracer, in TB granulomas of Mtb-infected macaques by a) serially imaging of infected macaques throughout disease with FDG and 64Cu-LLP2A; and b) injecting dye-LLP2A conjugates pre-necropsy to correlate immune cell localization by immunohistochemistry and flow cytometry with FDG imaging. Aim 2 will develop radiopharmaceuticals that specifically target macrophages by a) developing 18F-labeled small molecule antagonists of chemokine receptor type 2 (CCR2); and b) imaging Mtb-infected macaques with 18F-labeled mannose. In Aim 3 we will optimize neutrophil-specific probes based on 64Cu-labeled cFlFlF-based peptides by a) screening probes in a mouse model of inflammation to improve targeting and reduce clearance through the liver; and b) testing the uptake of radio- and dye-cFlFlF probes in neutrophils of Mtb-infected macaques. For Aim 4 we will investigate two to three of the most promising tracers developed in Aims 1-3 to monitor the cellular composition of TB lesions in macaques before, during, and after one chemotherapeutic (antibiotic) regimen, to determine if tracer uptake correlates with lesional histology and the efficiency of drugs to sterilize individual lesions.